Recombinant cage-like protein, method for producing the same, precious metal-recombinant cage-like protein complex, method for producing the same and recormbinant DNA

ABSTRACT

Using a gene recombination technique, a glutamic acid and an aspartic acid positioned in a channel of apoferritin are substituted with serine having a small size and no charges. Then, a glutamic acid positioned in a holding portion is substituted with a basic amino acid such as lysine or a neutral amino acid. Furthermore, at least one cysteine is introduced into the holding portion. This prevents a repulsive force due to electrostatic interaction between (AuCl 4 ) −  having a negative charge and a negative amino acid from occurring, which facilitates the capture of (AuCl 4 ) −  into the channel and the holding portion. The (AuCl 4 ) −  captured into the holding portion is subsequently reduced to Au, and thus apoferritin including gold particles can be produced.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to a precious metal-recombinantapoferritin complex produced with a gene recombination technique and amethod for producing the same, and techniques related thereto.

[0002] In recent years, in-depth research on bioelectronics, which is acombination of biotechnology and electronics, has been conducted, andsome products such as biosensors employing proteins such as enzymesalready have been put to practical use.

[0003] As one attempt to apply biotechnology to other fields, there isresearch in which fine particles made of metal or metal compounds areincorporated into apoferritin, which is a protein having the function ofholding metal compounds, to produce the fine particles having uniformsizes of nm order. Research to introduce various metals or metalcompounds suitable to the application of the fine particles intoapoferritin has been under way.

[0004] Hereinafter, apoferritin will be described. Apoferritin is aprotein that exists widely in the biological world and has the role ofregulating the amount of iron, which is an essential trace element inliving organisms. A complex of iron or an iron compound of apoferritinis called ferritin. If iron is present in an amount more than necessary,it is harmful to living organisms, so that excessive iron is stored inthe form of ferritin. The ferritin releases an iron ion as necessary andis converted back to apoferritin.

[0005]FIG. 1 is a schematic view showing the structure of ferritin(iron-apoferritin complex). As shown in FIG. 1, ferritin is a sphericalprotein having a molecular weight of about 460,000 in which 24 monomersubunits constituting one polypeptide chain are assembled bynon-covalent bonds, has a diameter of about 12 nm, and exhibits higherthermal stability and higher pH stability than those of common proteins.A hollow holding portion 4 having a diameter of about 6 nm is present inthe center of this spherical protein (outer shell 2), and the holdingportion 4 is connected to the outside via a channel 3. For example, whenincorporating a bivalent iron ion into ferritin, the iron ion enters itthrough the channel 3 and is oxidized in a site called “ferrooxidasecenter” in a subunit in a portion thereof, and then reaches the holdingportion 4 and is concentrated in a negative load region on the innersurface of the holding portion 4. Then, 3000 to 4000 iron atoms assembleand are held in the holding portion 4 in the form of ferrihydrite(5Fe₂O₃.9H₂O) crystal.

[0006] In this specification, a fine particle including a metal atomheld in the holding portion is referred to as a “core”. The diameter ofthe core 1 shown in FIG. 1 is substantially equal to the diameter of theholding portion 4, which is about 6 nm.

[0007] The core 1 can be removed by a comparatively simple chemicaloperation, and the particle constituted only by the outer shell 2without the core 1 is called apoferritin. Using apoferritin, anapoferritin-fine particle complex in which a metal or a metal compoundother than iron is supported artificially has been produced.

[0008] To date, it has been reported that metals such as manganese (P.Mackle, 1993, J. Amer. Chem. Soc. 115,8471-8472; F. C. Meldrum et al.,1995, J. Inorg. Biochem. 58, 59-68), uranium (J. F. Hainfeld, 1992,Proc. Natl. Acad. Sci. USA 89,11064-11068), beryllium (D. J. Price,1983, J. Biol. Chem. 258, 10873-10880), aluminum (J. Fleming, 1987,Proc. Natl. Acad. Sci. USA, 84, 7866-7870), zinc (D. Price and J. G.Joshi, Proc. Natl. Acad. Sci. USA, 1982, 79, 3116-3119), and cobalt (T.Douglas and V T. Stark, Inorg. Chem., 39, 2000, 1828-1830) or metalcompounds are introduced into apoferritin. The diameter of the core 1made of these metals or metal compounds is also substantially equal tothe diameter of the holding portion 4 of the apoferritin, which is about6 nm.

[0009] The process for forming the core 1 including an iron atom inferritin in the natural world proceeds generally in the followingmanner.

[0010] An amino acid having a negative charge at pH 7-8 is exposed ontothe surface of the channel 3 (see FIG. 1) for connecting the outside andthe inside of the ferritin particle, and a Fe²⁺ ion having a positivecharge is captured by the channel 3 by electrostatic interaction. Thechannels 3 are present in the number of 8 per apoferritin.

[0011] As on the inner surface of the channel 3, a large number ofglutamic acid residues, which are amino acid residues having a negativecharge at pH 7-8, are exposed onto the inner surface of the holdingportion 4 of the ferritin, and Fe²⁺ ions captured from the channel 3 areoxidized at the ferroxidase center and led to further inside of theholding portion 4. Then, the iron ions are concentrated by electrostaticinteraction and nucleus formation of a ferrihydrite (5Fe₂O₃.9H₂O)crystal occurs.

[0012] Thereafter, iron ions that are sequentially captured are attachedto the nucleus of this crystal, so that the nucleus made of iron oxideis grown and thus the core 1 having a diameter of 6 nm is formed in theholding portion 4. The capture of iron ions and the formation of thenucleus made of iron oxide are performed generally in the manner asdescribed above.

[0013] Next, an operation for introducing iron to apoferritin will bedescribed below.

[0014] First, a HEPES buffer solution, an apoferritin solution, and anammonium iron sulfate (Fe(NH₄)₂(SO₄)₂) solution are mixed in this orderto prepare a ferritin solution. In this ferritin solution, the finalconcentrations of the HEPES buffer solution, apoferritin and ammoniumiron sulfate are 100 mmol /L (pH 7.0), 0.5 mg/mL, and 5 mmol/L,respectively. All the operations for preparing ferritin are performed atroom temperature and stirring is performed with a stirrer.

[0015] Next, in order to complete a reaction for capturing iron ionsinto apoferritin and an oxidation reaction of the captured irons, theferritin solution is allowed to stand over night. This operationintroduces iron oxides having uniform sizes into the holding portion ofapoferritin, so that ferritin (a complex of apoferritin and a fineparticle) is produced.

[0016] Next, the ferritin solution is placed in a container, andcentrifuged at 3,000 rpm with a centrifugal separator for 15 to 30 minto remove a precipitate. Then, the resultant supernatant obtained afterthe precipitate is removed is centrifuged further at 10,000 rpm for 30min so as to precipitate an unwanted ferritin aggregate and remove it.At this point, ferritin is present in the supernatant in the form of adispersion.

[0017] Next, as the solvent of this supernatant, the 100 mmol/L HEPESbuffer solution of pH 7.0 is replaced by a 150 mmol/L NaCl solution bydialysis to prepare a new ferritin solution. Here, the pH does notnecessarily have to be adjusted.

[0018] Then, this ferritin solution is concentrated to an arbitraryconcentration between 1 and 10 mg/mL, and then CdSO₄ is added to thissolution such that the final concentration thereof becomes 10 mmol/L toaggregate the ferritin.

[0019] Next, the ferritin solution is centrifuged at 3,000 rpm for 20min to precipitate a ferritin aggregate in the solution. Thereafter, thebuffer component in the solution is replaced by a 10-50 mmol/L Trisbuffer solution of pH 8.0 containing 150 mmol/L NaCl by dialysis.

[0020] Next, the ferritin solution is concentrated and then is filtratedby gel filtration column to remove an aggregate of ferritin particles,so that discrete ferritin including iron oxide can be obtained.

[0021] The mechanism for capturing iron ions into ferritin and a methodfor preparing ferritin including iron oxide have been described above.Since all the other metal ions that have been reported so far to beintroduced are positive ions, it is believed that the capture of thesemetal ions to apoferritin substantially in the same mechanism as in thecase of iron ions. Therefore, the other ions basically can be introducedinto apoferritin substantially in the same operations as in the case ofiron ions.

[0022] Regarding apoferritin, the size of a particle that can be heldslightly varies with the type of the organism from which it is derived.Furthermore, there are spherical proteins that have similar structuresto that of apoferritin and can hold inorganic particles inside. Examplesthereof include Listeria ferritin derived from Listeria monocytogenesand Dps protein. There are proteins that are not spherical but can holdan inorganic particle similarly to ferritin, such as outer shellproteins of virus such as CCMV.

[0023] In the specification of the present application, proteins thatcan hold inorganic particles inside such as spherical proteins, outershell proteins of virus are referred to as “cage-like proteins”.

[0024] These cage-like proteins can hold inorganic particles includingiron.

[0025] Thus, ferritin holding a metal ion such as iron can be producedin the above-described method. However, since the inner surface of thechannel 3 of apoferritin and ferritin is positively charged as a whole,it is difficult to capture ions having the same negative charge intoapoferritin.

[0026] On the other hand, gold, platinum or the like cannot be ionizedalone in an aqueous solution, and only can be present as complex ions inan aqueous solution. Therefore, they are often used in the form ofnegative ions of chloroauric acid ions (AuCl₄)⁻ or (PtCl₄)²⁻.Consequently, it was difficult to capture precious metal atoms such asgold or platinum into apoferritin in the prior art.

SUMMARY OF THE INVENTION

[0027] Therefore, with the foregoing in mind, it is an object of thepresent invention to introduce precious metal atoms such as gold into acage-like protein such as apoferritin by modifying the inner structureof a cage-like protein such as apoferritin, and thus to form preciousmetal particles applicable to various microstructures.

[0028] A recombinant cage-like protein of the present invention isproduced by a gene recombination technique and includes a holdingportion that is present in an internal portion of the recombinantcage-like protein and can hold a precious metal particle; and atunnel-like channel for connecting the holding portion and an outside ofthe recombinant cage-like protein.

[0029] Thus, a precious metal particle having a uniform size ofnanometer order, can be formed in the holding portion of the recombinantcage-like protein, so that minute dot bodies made of a precious metalhaving excellent chemical stability by, for example, arranging preciousmetal-recombinant cage-like protein complexes on a substrate andremoving the protein portion. These dot bodies can be utilized, forexample, in a process for producing a semiconductor.

[0030] The recombinant cage-like protein is apoferritin, so that aprecious metal particle having a size of the nanometer order can beproduced efficiently.

[0031] The precious metal particle is gold or platinum, so that theformation of the dot bodies can be facilitated. The produced fineparticles can be applied to single-electron transistors or the like.

[0032] The recombinant cage-like protein includes a first neutral aminoacid that has a smaller molecular size than that of glutamic acid (Glu)and that of aspartic acid (Asp) in positions on an inner surface of thechannel in which a first glutamic acid and a first aspartic acid are tobe present, so that a repulsive force due to electrostatic interactionbetween precious metal complex ions having a negative charge and thechannel is prevented from occurring. As a result, the precious metalcomplex ions can be captured by the channel.

[0033] The first neutral amino acid is selected from the groupconsisting of serine, alanine, and glycine, so that precious metalcomplex ions can be captured by the channel without breaking thestereostructure of the recombinant cage-like protein.

[0034] The recombinant cage-like protein further includes a basic aminoacid or a second neutral amino acid in a position in the inner surfaceof the holding portion in which a second glutamic acid is to be present,so that a repulsive force due to electrostatic interaction betweenprecious metal complex ions having a negative charge and the holdingportion is prevented from occurring. In particular, when a basic aminoacid is provided in a position in which the second glutamic acid is tobe present, precious metal complex ions having negative charges arecaptured because of positive charges of this basic amino acid, so thatthe precious metal complex ions can be captured in a high concentrationby the holding portion.

[0035] The basic amino acid or the second neutral amino acid is selectedfrom the group consisting of arginine, lysine, and alanine, so thatprecious metal complex ions can be captured by the holding portionwithout breaking the stereostructure of the recombinant cage-likeprotein.

[0036] At least one cysteine substituted for an amino acid is present onthe inner surface of the holding portion, so that precious metal complexions captured by the holding portion can be reduced effectively, andprecious metal particles can be precipitated.

[0037] The recombinant cage-like protein includes a substance havingsmaller reduction function than that of cysteine in a position on theouter surface of the recombinant cage-like protein in which cysteine isto be present, so that the precious metal complex ions are preventedfrom being reduced on the outer surface of the recombinant cage-likeprotein. As a result, the precious metal particles are prevented frombeing precipitated on the outer surface of the recombinant cage-likeprotein, so that the yield of the precious metal-recombinant cage-likeprotein that holds a precious metal particle in the holding portion canbe increased.

[0038] A precious metal-recombinant cage-like protein complex of thepresent invention includes a holding portion that can hold a preciousmetal particle and a tunnel-like channel for connecting the holdingportion and the outside of the recombinant cage-like protein.

[0039] Thus, minute dot bodies made of the precious metal can be formedon a substrate by, for example, arranging the precious metal-recombinantcage-like protein complexes on a substrate and removing the proteinportion. These dot bodies can be utilized, for example, in a process forproducing a semiconductor.

[0040] The recombinant cage-like protein may be apoferritin.

[0041] The cage-like protein may hold a gold or platinum particle on theouter surface thereof.

[0042] The precious metal-recombinant cage-like protein complex includesa first neutral amino acid that has a smaller molecular size than thatof glutamic acid and that of aspartic acid in positions on the innersurface of the channel in which a first glutamic acid and a firstaspartic acid are to be present, so that a repulsive force due toelectrostatic interaction between precious metal complex ions having anegative charge and the channel is prevented from occurring. As aresult, the precious metal - recombinant cage-like protein complex canbe produced efficiently.

[0043] The first neutral amino acid can be selected from the groupconsisting of serine, alanine and glycine, so that the preciousmetal-recombinant cage-like protein complex can be formed without thestereostructure.

[0044] The precious metal-recombinant cage-like protein complex furtherincludes a basic amino acid or a second neutral amino acid in a positionin the inner surface of the holding portion in which a second glutamicacid is to be present, so that a repulsive force due to electrostaticinteraction between precious metal complex ions having a negative chargeand the holding portion is prevented from occurring. As a result, theprecious metal—recombinant cage-like protein complex can be producedefficiently.

[0045] The basic amino acid or the second neutral amino acid is selectedfrom the group consisting of arginine, lysine, and alanine, so that theprecious metal particles can be held without breaking thestereostructure.

[0046] At least one cysteine substituted for an amino acid is present onthe inner surface of the holding portion, so that the preciousmetal-recombinant cage-like protein complex can be formed easily in asolution containing precious metal ions.

[0047] A recombinant DNA of the present invention encodes an amino acidsequence of a recombinant cage-like protein including a holding portionthat can hold a precious metal particle and a tunnel-like channel forconnecting the holding portion and the outside of the recombinantcage-like protein.

[0048] This recombinant DNA makes it possible to mass-produce therecombinant cage-like protein using a protein engineering technique.

[0049] The recombinant cage-like protein may be apoferritin.

[0050] The precious metal particle may be gold or platinum.

[0051] The recombinant DNA includes a first neutral amino acid that hasa smaller molecular size than that of glutamic acid and that of asparticacid in positions on the inner surface of the channel in which a firstglutamic acid and a first aspartic acid are to be present, so that therecombinant protein can be obtained easily.

[0052] The first neutral amino acid is selected from the groupconsisting of serine, alanine and glycine, so that a large amount of therecombinant cage-like protein to form precious metal particlesefficiently in the holding portion can be obtained.

[0053] The recombinant DNA further includes a basic amino acid or asecond neutral amino acid in a position on the inner surface of theholding portion in which a second glutamic acid is to be present, sothat a large amount of homogeneous recombinant cage-like protein to formprecious metal particles efficiently in the holding portion can beobtained.

[0054] The basic amino acid or the second neutral amino acid is selectedfrom the group consisting of arginine, lysine, and alanine, so thatrecombinant cage-like protein that can hold precious metal particlesefficiently can be obtained easily.

[0055] At least one cysteine substituted for an amino acid is present onthe inner surface of the holding portion, so that recombinant cage-likeprotein that can hold precious metal particles more efficiently can beobtained easily.

[0056] A method for producing a recombinant cage-like protein of thepresent invention includes the step (a) of substituting a first glutamicacid and a first aspartic acid that are positioned on the inner surfaceof a channel with a first neutral amino acid having a smaller molecularsize than that of glutamic acid and that of aspartic acid.

[0057] This method makes it possible to easily produce the recombinantcage-like protein that can capture precious metal particles into thechannel efficiently.

[0058] The cage-like protein may be apoferritin.

[0059] In the step (a), the first neutral amino acid is selected fromthe group consisting of serine, alanine, and glycine, so that it ispossible to produce a recombinant cage-like protein that can captureprecious metal complex ions into the channel without breaking thestereostructure of the recombinant cage-like protein.

[0060] The method for producing a recombinant cage-like protein furtherincludes the step (b) of substituting a second glutamic acid present onthe inner surface of the holding portion that is inside the recombinantcage-like protein with a basic amino acid or a second neutral aminoacid, so that the recombinant cage-like protein that can captureprecious metal particles into the holding portion efficiently can beproduced easily.

[0061] In the step (b), the basic amino acid or the second neutral aminoacid is selected from the group consisting of arginine, lysine andalanine, so that precious metal complex ions can be captured into theholding portion without breaking the stereostructure of the recombinantcage-like protein.

[0062] The method for producing a recombinant cage-like protein furtherincludes the step (c) of substituting at least one amino acid positionedon the inner surface of the holding portion with cysteine, so that arecombinant cage-like protein that allows precious metal complex ionscaptured into the holding portion to be reduced effectively toprecipitate precious metal particles. When the precious metal complexions are reduced, the molecular size is decreased, so that the captureof the precious metal complex ions into the holding portion can bepromoted.

[0063] The method for producing a recombinant cage-like protein furtherincludes the step (d) of replacing at least one cysteine positioned onthe outer surface of the recombinant cage-like protein by a substancehaving a smaller reduction function than that of cysteine, so that arecombinant cage-like protein in which the reduction of the preciousmetal complex ions on the outer surface is suppressed can be produced.

[0064] A method for producing a precious metal-recombinant cage-likeprotein complex includes the steps: (a) mixing a precious metal complexion solution and a recombinant cage-like protein solution to form aprecious metal-recombinant cage-like protein complex, and (b) passing asolution containing the precious metal-recombinant cage-like proteincomplex prepared in the step (a) through a gel filtration column topurify the precious metal-recombinant cage-like protein complex.

[0065] This method makes it possible to fractionate the recombinantcage-like protein holding precious metal on the outer surface, therecombinant cage-like protein including the precious metal, and a sidereaction product or the like from each other by the size, so that adesired purified precious metal-recombinant cage-like protein complexcan be selected.

[0066] The precious metal in the step (a) is gold or platinum, so thatas described above, the dot bodies made of gold or platinum to beutilized in, for example, a production process of a semiconductor deviceor the like can be formed, and in this process, the reduction process ofthe dot bodies, which was conventionally necessary, can be eliminated.

BRIEF DESCRIPTION OF THE DRAWINGS

[0067]FIG. 1 is a schematic view showing the structure of ferritin.

[0068]FIGS. 2A to 2C are schematic cross-sectional views of recombinantapoferritin according to a first embodiment of the present invention.

[0069]FIG. 3 is a schematic view of gold-including apoferritin arrangedon a substrate.

[0070]FIG. 4 is an electron micrograph of the gold-including apoferritinarranged on a substrate.

[0071]FIG. 5 is a schematic view showing a nucleotide detector accordingto a second embodiment of the present invention.

[0072]FIG. 6 is a cross-sectional view showing the structure of anon-volatile memory cell in which dot bodies made of gold particles areused for a floating gate according to a third embodiment of the presentinvention.

[0073]FIGS. 7A to 7C are cross-sectional views showing a process forforming a microstructure according to a fourth embodiment of the presentinvention.

[0074]FIG. 8 is a cross-sectional view of an optical semiconductordevice according to a fifth embodiment, which utilizes themicrostructure formed in the fourth embodiment.

[0075]FIG. 9 is a schematic view showing a gold-apoferritin complexholding precious metal particles both on the outer surface and in theholding portion.

DETAILED DESCRIPTION OF THE INVENTION First Embodiment

[0076] A first embodiment of the present invention will be describedbelow.

Production of Recombinant Apoferritin

[0077] The inventors of the present invention considered the followingtwo aspects to be primary detriments to the introduction of gold (Au)atoms into apoferritin.

[0078] One is electrostatic interaction between a chloroauric acid ion(AuCl₄)⁻ and apoferritin. The amino acids that have negative charges,such as glutamic acid or aspartic acid, are exposed onto the innersurface of the channel 3 and the inner surface of the holding portion 4of ferritin (apoferritin-iron complex) shown in FIG. 1. The capture of(AuCl₄)⁻, which is a negative ion, into apoferritin is inhibited by anelectrostatic interaction with these amino acids having negative ions.

[0079] Another detriment is that the size of (AuCl₄)⁻ is larger thanthat of an iron ion. For this reason, unless the size of the channel 3of the apoferritin is increased, it is physically difficult to capture(AuCl₄)⁻ into the channel 3.

[0080] In order to solve these problems, the inventors modifiedapoferritin with a technique of gene recombination in the followingmanner. Hereinafter, in this specification, “recombinant apoferritin”refers to apoferritin to which a variation is introduced with a generecombination technique. When a site of an amino acid residue isspecified in this specification, it means, unless otherwise, a site ofapoferritin derived from equine liver to which no variation isintroduced. Since apoferritin is constituted with 24 monomer subunits,“the amino acid sequence of apoferritin” means the amino acid sequenceof the monomer subunits.

[0081] The gene sequence encoding apoferritin derived from equine liverand the amino acid sequence of apoferritin are known, and thestereostructure thereof has been clarified. The monomer of apoferritinis constituted with 175 amino acid residues. Among these, the 128^(th)amino acid residue of aspartic acid (Asp) and the 131^(st) amino acidresidue of glutamic acid (Glu) are both positioned on the inner surfaceof the channel 3, and the 58^(th), 61^(st) and 64^(th) amino acidresidues of glutamic acids are all positioned on the inner surface ofthe holding portion 4. The first to eighth amino acid residues aredeleted by in vivo processing.

[0082] Next, the electrostatic interaction in apoferritin will bedescribed.

[0083] As described above, aspartic acid and glutamic acid havingnegative charges are located on the inner surfaces of the holdingportion 4 and the channel 3 of apoferritin in a neutral solution, sothat the potential Vin of the entire inner surface of the apoferritin islower than the potential Vout of the outside of the apoferritin. Morespecifically, the difference ΔV in the potential between the inside andthe outside of the apoferritin defined as

ΔV=Vin−Vout is ΔV<0(mV).

[0084] Here, since (AuCl₄)⁻ has a negative charge, it is known that therelationship between Cin, Cout and ΔV is expressed by the followingequation (1), where Cin is the concentration of (AuCl₄)⁻ inside theapoferritin, and Cout is the concentration of (AuCl₄)⁻ in the solution.

Cout/Cin=e ^(−ΔV/kT)  (1)

[0085] where e is a natural logarithm, k is Boltzmann logarithm, and Tis an absolute temperature. This equation indicates that when thetemperature is constant, the (AuCl₄)⁻ concentration inside theapoferritin can be increased exponentially by increasing ΔV. Forexample, when ΔV is a positive value and ΔV is increased by a factor of4, Cout/Cin is about 80.

[0086] On the other hand, the reduction reaction of (AuCl₄)⁻ → u in theinner surface of the apoferritin is accelerated as the concentrationinside the apoferritin increases.

[0087] Considering the above conditions, the inventors of the presentinvention concluded that it is necessary that Cin is at least threetimes larger than Cout in order to produce gold particles efficiently inthe holding portion 4 of the apoferritin in a solution. ΔV thatsatisfies this condition at room temperature is about 25 (mV) or more.In particular, in order to produce gold particles in the holding portion4 at a sufficient speed, it seems to be preferable that ΔV is about 100(mV) or more.

[0088] Herein, ΔV can be obtained by adding all the charges of the basespresent on the inner surface of the apoferritin, taking the positioninto consideration. For example, three of the glutamic acids positionedin the holding portion 4 is substituted with lysine (Lys) in anapoferritin monomer, so that ΔV in the apoferritin is calculated to beabout 200 mV This is believed to be a sufficient potential difference toproduce gold particles in the holding portion 4 of the apoferritin.

[0089] The inventors of the present invention produced a recombinantapoferritin as follows that can hold gold particles, based on the abovecalculations.

[0090]FIGS. 2A to 2C are schematic views showing the structure of arecombinant apoferritin produced based on the above findings.

[0091] First, FIG. 2A shows apoferritin derived from equine liver(hereinafter, referred to as “apoferritin”) in which the 128^(th) aminoacid of aspartic acid (Asp) and the 131st amino acid of glutamic acid(Glu) are substituted with serine (Ser). Even if aspartic acid orglutamic acid is substituted with serine, the stereostructure of theapoferritin can be maintained. The first to the eight amino acids in theapoferritin are projected from the outer surface of the apoferritin, andmay cause a problem in producing a higher order structure such astwo-dimensional crystallization, so that they are deleted. Thisrecombinant apoferritin is expressed as “fer-8-ser” in the following.

[0092] The substitution of aspartic acid and glutamic acid havingnegative charges present on the inner surface of the channel 3 withserine having no charge eliminates electrostatic repulsion, which makesit easier to capture (AuCl₄)⁻ (7 in FIG. 2A) having a negative chargeinto the channel 3. Furthermore, since the serine residue has a smallersize than that of the aspartic acid residue or the glutamic acidresidue, so that the physical detriment to the capture of (AuCl₄)⁻ intothe channel is smaller.

[0093] Next, FIG. 2B shows a recombinant apoferritin in which the58^(th), 61^(st) and 64^(th) amino acids of glutamic acids in the aminoacid sequence of fer-8-Ser are each substituted with arginine (Arg).This recombinant apoferritin is expressed as “fer-8-Ser-Arg” in thefollowing.

[0094] The substitution of the 58^(th), 61^(st) and 64^(th) amino acidsof glutamic acids present on the inner surface of the holding portion 4of the apoferritin with arginine having a positive charge makes itpossible to guide (AuCl₄)⁻ captured by the channel 3 to the holdingportion 4 of the apoferritin. In this case, even if the glutamic acid issubstituted with arginine, the stereostructure of the apoferritin can bemaintained. The (AuCl₄)⁻ 7 guided to the holding portion 4 aresequentially reduced to gold (Au) atoms 7′. As the amino acid that issubstituted with the 58^(th), 61^(st) and 64^(th) amino acids ofglutamic acids, any amino acid that has no negative charge can be used,and Lys, which is a basic amino acid, Ala, which is a nonpolar aminoacid, and a neutral amino acid can be used.

[0095] Next, FIG. 2C shows a recombinant apoferritin in which the54^(th) amino acid of glutamic acid and the 65^(th) amino acid ofarginine in the amino acid sequence of fer-8-Ser-Arg are substitutedwith cysteine (Cys). This recombinant apoferritin is expressed as“fer-8-Ser-Arg-Cys” in the following.

[0096] The 54^(th) amino acid of glutamic acid and the 65^(th) aminoacid of arginine in the amino acid sequence of fer-8-Ser-Arg are presenton the inner surface of the holding portion 4 of apoferritin, so thatthe substitution of these amino acids with cysteine makes it possible toreduce the (AuCl₄)⁻ 7 captured by the holding portion 4 so that goldfine particles can be precipitated. Thus, the core 1 made of gold can beformed in the holding portion 4 by the operations described later.

[0097] To produce the above-described recombinant apoferritin, a knowngene recombination technique and a method for expressing a protein areused in the manner as described below.

[0098] First, a DNA fragment that encodes the amino acid sequence ofapoferritin is cut out with a suitable restriction enzyme from a plasmidTakeda99224 (see S. Takeda et al. Biochim. Biophys. Acta., 1174,218-220,1993) produced by Takeda et al. in which DNA of apoferritin derived fromequine liver is incorporated.

[0099] Next, this DNA fragment is inserted into pMK-2, which is avector-plasmid for expressing a protein, to produce a plasmid forexpressing apoferritin.

[0100] Then, PCR (polymerase chain reaction) is performed, using thisplasmid for expressing apoferritin as the template and a single strandDNA fragment in which a desired variation is incorporated as the primer,so that the desired variation is introduced site-specifically to adesired position of the DNA encoding the amino acids of apoferritin.Thus, a plasmid including a DNA fragment of a variant apoferritin genein which DNA in a portion encoding the first to the eighth amino acidsof the apoferritin is deleted is produced. The DNA fragment of thisapoferritin gene may cut out and incorporated into anothervector-plasmid, if necessary.

[0101] Then, the produced plasmid is introduced into a commerciallyavailable E. coli (e.g., Nova Blue) and transformed, and then this E.coli is cultured in a large amount at 37° C. with a jar fermentor (massculturing apparatus). The transformed E. coli has resistance toampicilline, which is used as an indicator to distinguish it from E.coli that has not been transformed, so that the transformed E. coli canbe screened.

[0102] In this E. coli, the DNA of the recombinant apoferritinincorporated in the plasmid expresses, and apoferritin in which thefirst to the eight amino acid residues are deleted (hereinafter,referred to as “fer-8”) has been mass-produced. fer-8 is extracted andpurified from the E. coli bacteria in the manner described later.

[0103] Next, in order to produce fer-8-Ser, PCR is performed, using theplasmid obtained in the above-described operation to which DNA encodingthe amino acid sequence of fer-8 is incorporated as the template and asingle strand DNA fragment encoding the amino acid sequence in which the128^(th) amino acid of aspartic acid and the 131^(st) amino acid ofglutamic acid of apoferritin are substituted with serine as the primer.

[0104] Then, a plasmid to which DNA encoding the amino acid sequence offer-8-Ser is inserted is produced in the same manner as for productionof fer-8, and this plasmid is introduced into E. coli (Nova Blue) andtransformed, and then the transformed E. coli is cultured in a largeamount, and then fer-8-Ser is extracted and purified from the E. colibacteria in the manner described later.

[0105] Then, a plasmid to which DNA encoding the amino acid sequence offer-8-Ser-Arg is inserted and fer-8-Ser-Arg are obtained, and then aplasmid to which DNA encoding the amino acid sequence offer-8-Ser-Arg-Cys is inserted and fer-8-Ser-Arg-Cys are obtained in thesame manner as above.

[0106] The procedure for extracting and purifying the variantapoferritin in the above operations is as follows.

[0107] First, a culture liquid of E. coli that has been cultured istransferred to a centrifugal tube, set in a centrifugal separator, andcentrifuged at 4° C. and 10,000 rpm for 25 min to precipitate the E.coli bacteria.

[0108] Next, after the precipitated bacteria are collected, the bacteriaare disintegrated in a liquid with an ultrasonic disintegrator so thatapoferritin is eluted in the liquid. Then, the liquid in which thebacteria are disintegrated is transferred to a centrifugal tube, set ina centrifugal separator, and centrifuged at 4° C. and 10,000 rpm for 25min to precipitate the bacteria that has been left undisintegrated.

[0109] Next, a supernatant is collected from the centrifugal tube, andthe supernatant is subjected to a heat treatment at 60° C. for 15 min,and then transferred to a centrifugal tube for centrifugation at 4° C.and 10,000 rpm for 25 min. This operation modifies unwanted protein,which precipitates in the bottom of the tube.

[0110] Then, after a supernatant is collected from the centrifugal tube,column chromatography is performed with Q-sepharose HP (gel filtrationcolumn) at 4° C. to collect an apoferritin fraction contained in thesupernatant. This apoferritin fraction is further passed throughSephacryl S-300 (gel filtration column) at 25° C. for columnchromatography so as to be purified. This operation removes impuritiesand a purified recombinant apoferritin can be obtained.

[0111] In the present invention, when DNA encoding a modifiedapoferritin can be obtained, this DNA can be amplified by a knowntechnique. Therefore, for mass production of recombinant apoferritin,there is no need to perform a process of recombination of genes again.

Production of Apoferritin holding Gold Particles

[0112] First, a recombinant apoferritin solution and a KAuCl₄ solution(or HAuCl₄) solution are mixed to prepare a solution in which the finalconcentrations of the recombinant apoferritin and KAuCl₄ are 0.5 mg/mLand 3 mmol /L, respectively and the pH is 7 to 9. Thereafter, thesolution is allowed to stand at room temperature for at least 24 hoursso that gold particles are captured into the inside of the apoferritinto form a gold-apoferritin complex. As a buffer, when the pH is 7 to 8,100 mM phosphoric acid is preferably used, and when the pH is 8 to 9,Tris-HCl is preferably used

[0113] At this point, if either one of adding NaBH₄ to the solution suchthat the concentration thereof is 1 mM or less, adding alcohol such asethanol to the solution such that the concentration thereof is 10% orless (v/v), or irradiating the solution with light or UV rays, it ispossible to accelerate the reduction reaction of (AuCl₄)⁻ so that thereaction time can be shortened. However, if the concentration of NaBH₄is more than 1 mM, or if the concentration of ethanol is more than 10%(v/v), (AuCl₄)⁻ is reduced before being captured into the inside of theapoferritin, so that gold particles may be precipitated on the outersurface of the apoferritin. The size of the gold particles precipitatedon the outer surface of the apoferritin is non-uniform to a largerextent than the size of gold particles formed in the holding portion 4of the apoferritin.

[0114] Inside the apoferritin, the surface of the precipitated goldparticles itself catalyzes the reduction reaction of (AuCl₄)⁻(autocatalytic function). Thus, the reduction reaction of (AuCl₄) ⁻continues until the holding portion 4 of the apoferritin is filled.

[0115] The pH of the solution is set to 7 to 9 for the followingreasons. If the pH is 6 or less, the reduction of (AuCl₄)⁻ hardlyoccurs, and if the pH is 10 or more, the progress of the reduction of(AuCl₄)⁻ cannot be controlled.

[0116] Thereafter, side reaction products and apoferritin that does nothold gold particles are removed in the same manner as when purifyingferritin including iron inside, and the resultant solution isfractionated by gel column chromatography so that the apoferritinincluding gold particle inside is collected in the form of a solution.In this case, apoferritin in which gold particles are formed, not in theholding portion 4, but on its outer surface and a small amount ofapoferritin in which gold particles are formed both in the holdingportion 4 and on the outer surface can be obtained at the same time.

[0117] If fer-8-Ser and fer-8-Arg are used as recombinant apoferritin ina reaction to capture gold particles into apoferritin, apoferritin inwhich gold particles are formed on its outer surface is also generatedas well as apoferritin including gold particles inside. This seems to bebecause the speed of a reaction to precipitate gold on the outer surfaceof apoferritin is faster than that of a reaction to form gold particlesin the holding portion 4 of the apoferritin.

[0118] On the other hand, if fer-8-Ser-Arg is used as a recombinantapoferritin, the yield of the apoferritin including gold particlesinside is improved significantly. This is because the reduction reactionof (AuCl₄)⁻ in the holding portion 4 of the apoferritin is acceleratedby the reductive function of cysteine (Cys) introduced into the holdingportion 4. The diameter of gold particles included inside theapoferritin is uniformly about 6 nm. In other words, gold particleshaving a uniform size of the nanometer order can be formed efficientlyby using a recombinant apoferritin produced in this embodiment,fer-8-Ser-Arg-Cys. Fine gold particles have applications or advantagesthat other metals do not have, such as an application to a DNA sensor.

[0119] In this embodiment, the apoferritin derived from equine liver isused, but apoferritin derived from other organs or other livingorganisms, that is, proteins made of monomer subunit polymer andincluding a holding portion inside can be used. Apoferritin derived fromsome other living organisms such as Listeria ferritin derived fromListeria monocytogenes have a stereostructure similar to that of theapoferritin derived from equine, so that a recombinant apoferritin canbe obtained in the same operation. The diameter of the core of ametal-apoferritin complex is slightly different, depending on the type,so that the diameter of gold particles can have a variation. In additionto that, a cage-like protein, which can hold a metal or the like insidecan hold gold particles by changing the charge of the channel and theinside as done in this embodiment.

[0120] Furthermore, in the case of proteins of other ferritin familiessuch as a Dps protein constituted with 12 monomer subunits and includingan inorganic substance inside, precious metal particles can be held withthe same gene recombination technique as in the case of apoferritin.

[0121] In this embodiment, the 128^(th) amino acid of aspartic acid andthe 131^(st) amino acid of glutamic acid present on the inner surface ofthe channel 3 of apoferritin are both substituted with serine. However,instead of serine, they can be substituted with glycine or alanine,which is a neutral amino acid having an even smaller molecular weight.

[0122] In this embodiment, fer-8-Ser-Arg is used as a recombinantapoferritin, but basic, or non-polar or neutral amino acids having nonegative changes, such as lysine or alanine, can be used to substitutethe 58^(th), 61^(st), and 64^(th) amino acids of glutamic acids in theamino acid sequence of the apoferritin. A recombinant apoferritin inwhich the 58^(th), 61^(st), and 64^(th) amino acids of glutamic acids inthe amino acid sequence are substituted with lysine is represented byfer-8-Ser-Lys in the following.

[0123] A recombinant apoferritin in which the 58^(th), 61^(St), and64^(th) amino acids of glutamic acids in the amino acid sequence aresubstituted with alanine is represented by fer-8-Ser-Ala.

[0124] A recombinant apoferritin in which the 54^(th) amino acid ofglutamic acid and the 65^(th) amino acid of arginine in fer-8-Ser-Lysare both substituted with cysteine is represented by fer-8-Ser-Lys-Cys.A recombinant apoferritin in which the 54^(th) amino acid of glutamicacid and the 65^(th) amino acid of arginine in fer-8-Ser-Ala are bothsubstituted with cysteine is represented by fer-8-Ser-Ala-Cys.

[0125] Among these, the DNA sequence encoding the amino acid sequence offer-8-Ser-Lys-Cys is described in sequence 1, and the amino acidsequence of fer-8-Ser-Lys-Cys is described in sequence 2 in the sequencelisting. The amino acid of sequence 2 starts with the 9^(th) amino acid,tyrosine.

[0126] In fer-8-Ser-Lys-Cys produced in this embodiment, the DNAsequence encoding Lys of the 58^(th), 61^(st) and 64^(th) amino acids(50^(th), 53^(rd), and 56^(th) amino acids in the sequence 2) of Lys are“aag”, but this can be “aaa” encoding Lys, instead. For Ser of the128^(th) and 131^(st) amino acids (120^(th) and 123^(rd) amino acids inthe sequence 2) or Cys of the 54^(th) and 65^(th) amino acids (46th and57^(th) amino acids in the sequence 2), other sequences than those shownin the sequence 1 can be used, as long as it is a DNA sequence encodingthese amino acids. This is true for other recombinant apoferritin.

[0127] The 127^(th) amino acid of cysteine of the recombinantapoferritin produced in this embodiment such as fer-8-Ser-Arg-Cys,fer-8-Ser-Lys-Cys and fer-8-Ser-Ala-Cys is positioned on the outersurface of the apoferritin, and it is estimated that this cysteineprecipitates gold particles on the outer surface of the apoferritin.Therefore, when the 127^(th) amino acid of cysteine offer-8-Ser-Arg-Cys, fer-8-Ser-Lys-Cys and fer-8-Ser-Ala-Cys issubstituted with a substance having a smaller reduction function thanthat of the cysteine, gold particles are suppressed from beingprecipitated on the outer surface of the apoferritin, and the yield ofthe apoferritin including gold particles inside can be improved further.In order to achieve this, the 127^(th) amino acid of cysteine may besubstituted with an amino acid such as alanine, or may be reacted withchemicals that react with a cysteine reside to suppress the reductionfunction.

[0128] In this embodiment, a gold-apoferritin complex is produced, butinstead of introducing (AuCl₄)⁻ to apoferritin, chloroplatinic acid(PtCl₄)²⁻ is introduced into a recombinant apoferritin to produceapoferritin holding platinum particles. However, since (PtCl₄)²⁻ iseasily reduced in a solution of pH 7 to 9 so that platinum isprecipitated in the solution, it is necessary that the pH of thesolution is lower than 7. In this case, 100 mM acetic acid is used as abuffer when pH is about 4, and 100 mm β-alanine is used when pH is about3.

[0129] An example of industrial application of the recombinantapoferritin holding precious metal produced in this embodiment will bedescribed in the following embodiment.

Second Embodiment

[0130] First, the structure of the nucleotide detector in thisembodiment will be described. FIG. 5 is a cross-sectional view showingthe structure of the nucleotide detector in this embodiment.

[0131] As shown in FIG. 5, the nucleotide detector 10 in this embodimentis a DNA sensor, and includes a substrate 11, gold particles 12 having ananometer size (a diameter of about 6 nm) arranged on the surface of thesubstrate 11 with high density and high precision (with a gap of about12 nm between neighboring particles), and single stranded DNAs (thiolDNAs) 13 having a sulfur atom at their ends, and the gold particles 12are bonded to the thiol DNAs 13.

[0132] Next, a method for producing the nucleotide detector 10 in thisembodiment will be described. In order to produce the nucleotidedetector 10 in this embodiment, the gold particles 12 having a diameterof about 6 nm should be arranged and fixed two-dimensionally with highdensity and high precision on the surface of the substrate 11.

[0133] First, the recombinant apoferritin holding the gold particles 12of the first embodiment (a complex of fer-8-Ser-Arg-Cys and goldparticles; referred to as gold-including apoferritin 15 in thefollowing) are arranged on the surface of the substrate 11 in a methodas described below.

[0134]FIG. 3 is a schematic view showing the gold-including apoferritin15 arranged on the substrate 11, and FIG. 4 is an electron micrograph ofthe gold-including apoferritin 15 arranged on the substrate 11.

[0135] For imaging of the electron micrograph of FIG. 4, gold glucosehaving a size that is large enough not to be captured into apoferritinis used for staining. Gold glucose staining is used because whenstaining is performed with a regular pigment, the pigment enters theapoferritin so that the presence of a gold particle cannot be confirmed.

[0136] This operation forms a film of the gold-including apoferritin 15arranged with high density and high precision as shown in FIG. 3 on thesubstrate 11. FIG. 4 indicates that the outer diameter of theapoferritin is about 12 nm.

[0137] Then, the outer shell 2 made of protein of the gold-includingapoferritin 15 is removed so that only the gold particles 12 are left.Then, the thiol DNAs 13 are bonded to the gold particles 12. The DNAsused here are single stranded DNAs.

[0138] In this embodiment, a known method can be used to arrange and fixthe gold-including apoferritin 15 two-dimensionally with high densityand high precision on the surface of the substrate 11.

[0139] For example, the transfer method (Adv. Biophys., Vol. 34, p99-107(1997)) that has been developed by Yoshimura et al, which will bedescribed below, can be used.

[0140] In this method, first, a liquid in which the gold-includingapoferritin 15 is dispersed is injected to a 2% sucrose solution with asyringe. Then, the liquid comes up toward the liquid surface of thesucrose solution.

[0141] Then, the liquid that has first reached the interface between airand the liquid forms an amorphous film made of modified apoferritin, andthe liquid that has reached later is attached under the amorphous film.

[0142] Then, two-dimensional crystal of the gold-including apoferritin15 is formed under the amorphous film. Then, the substrate 11 (siliconwafer, carbon grid, glass substrate or the like) is placed on the filmconsisting of the amorphous film and the two-dimensional crystal of thegold-including apoferritin 15, so that the film including thegold-including apoferritin 15 is transferred onto the surface of thesubstrate 11.

[0143] This method makes it possible to arrange the gold-includingapoferritin 15 on the substrate 11 with high density and high precision,as shown in FIG. 3.

[0144] In this case, if the surface of the substrate 113 is treated soas to be hydrophobic, the film can be transferred onto the surface ofthe substrate 11 more easily.

[0145] Next, the outer shell 2 made of protein is removed. Proteinmolecules are generally weak to heat, so that the outer shell 2 can beremoved by a heat treatment as described below.

[0146] For example, when the substrate 11 with the gold-includingapoferritin 15 is left undisturbed in an inert gas such as nitrogen at400 to 500° C. for about one hour, the outer shell 2 and the amorphousfilm made of protein are burned out, so that the gold particles 12remain on the substrate 11 in the form of dots that are arrangedregularly in a two-dimension with high density and high precision.

[0147] As described above, the gold particles 12 held in thegold-including apoferritin 15 are allowed to appear on the substrate 11two-dimensionally and arranged with high density and high precision.

[0148] Next, formation of the nucleotide detector 10 of this embodimentwill be described below.

[0149] The nucleotide detector 10 of this embodiment is obtained bybonding thiol DNAs 13 to the gold particles 12 arranged on the substrate11 in the manner as described above.

[0150] The gold particles 12 can be bonded to the thiol DNAs 13 simplyby bringing the substrate 11 in which the gold particles 12 are arrangedinto contact with an aqueous solution of the thiol DNAs 13 and leavingas it is for a predetermined time. This bonding can be achieved becausesulfur easily reacts with gold and thus easily forms a covalent bondwith the gold particles 12 at the end of the thiol DNA 13 or thiol RNA.

[0151] More specifically, when the thiol DNAs 13 in the aqueous solutionare brought into contact with the gold particles 12 on the substrate 11,sulfur atoms S of the thiol DNAs 13 are covalently bonded to the goldparticles 12 in the one-to-one correspondence manner, so that the thiolDNAs 13 are arranged on the substrate 11 with very high density and highprecision. Since the gold particles 12 on the substrate 11 are arrangedtwo-dimensionally with very high density and high precision, the thiolDNAs 13 bonded to the gold particles 12 are also arrangedtwo-dimensionally with high density and high precision, so that in thenucleotide detector 10, particles are arranged uniformly in the numberper unit in accordance with the size of the particles.

[0152] In this process, instead of the thiol DNAs 13, thiol RNAs ornucleotides such as PCR primer whose end is thiolized can be used.

[0153] In the above process, the concentration of the thiol DNAs 13 inthe aqueous solution can be theoretically such that the number of thegold particles 12 on the substrate 11 matches the number of thiol DNAs13. However, in reality, it is preferable that the number of the thiolDNAs 13 is larger than that of gold particles 12. Therefore, in thisembodiment, an aqueous solution including a high concentration of thethiol DNAs 13 is used so that the thiol DNAs 13 are contained in thenumber of molecules of more than that of the gold-including apoferritin15 that is contained in the liquid in the form of a dispersion.

[0154] Furthermore, as the temperature of the aqueous solution of thethiol DNAs 13 is higher, the bonding between the sulfur atoms S of thethiol DNAs 13 and the gold particles 12 is promoted. However, if thetemperature is too high, it becomes difficult to handle the thiol DNAs13, for example, due to a large convection current. Furthermore, toohigh temperatures are also disadvantageous in view of energyconsumption, so that in general, it is preferable to heat the aqueoussolution of the thiol DNAs 13 to about 20 to 60° C. for theabove-described process.

[0155] Thus, the nucleotide detector 10 of this embodiment that iscapable of easily detecting DNA or RNA to be detected can be obtained.

[0156] Next, a method for detecting DNA when the nucleotide detector 10is used as a DNA sensor will be described.

[0157] First, a solution containing a DNA group to be subjected todetection (DNA group to be detected) is prepared and the DNA group to bedetected has been subjected to a fluorescent-labeling treatmentbeforehand.

[0158] The solution of the fluorescent-labeled DNA group to be detectedis brought into contact with the nucleotide detector 10 in which thethiol DNAs 13 are arranged and left undisturbed.

[0159] After a predetermine period of time has passed, when there is aDNA hybridized with the thiol DNA 13 of the nucleotide detector 10 in agroup of DNAs to be detected, the thiol DNA 13 of the nucleotidedetector 10 and the DNA in the group of DNAs to be detected constitute adouble helix and establish a stable bond.

[0160] Next, if the nucleotide detector 10 is washed with a solutionfree from a phosphor, such as water, the DNA that is not bonded to thethiol DNA 13 of the nucleotide detector in the group of DNAs to bedetected and a trace amount of phosphors left on the nucleotide detector10 can be removed.

[0161] Thereafter, fluorescence is observed by irradiating the surfaceof the nucleotide detector 10 with a light source such as laser. At thispoint, if there is a DNA having a sequence that is hybridized with thethiol DNA 13 of the nucleotide detector 10 in the group of DNAs to bedetected, fluorescence occurs.

[0162] As described above, whether or not there is a DNA having apredetermined sequence in the group of DNAs to be detected can bedetected by detecting whether or not fluorescence occurs.

[0163] In particular, in the nucleotide detector 10 of this embodiment,the thiol DNAs are arranged with high density and high precisionuniformly over the entire substrate. Therefore, the intensity offluorescence is high, and the fluorescence occurs highly precisely anduniformly, so the nucleotide detector 10 of this embodiment can be usedas a high performance DNA sensor having a very high SN ratio. Therefore,when the nucleotide detector 10 of this embodiment is used as a DNAsensor and a fluorescence intensity higher than a predetermined value isobtained, it is determined that a DNA having a predetermined sequence ispresent in the group of DNAs to be detected. That is to say, there isalmost no possibility of erring in the determination of the presence ofthe DNA having a predetermined sequence.

[0164] Furthermore, in the nucleotide detector 10 of this embodiment,the thiol DNAs are arranged with high density and high precisionuniformly over the entire substrate, and there is almost no possibilitythat the fluorescence intensity after the hybridization of the DNAhaving a predetermined sequence differs from substrate to substrate.Therefore, there is no need of changing the setting of a threshold ofthe fluorescence intensity for each substrate in order to determine thepresence of hybridized DNAs, which reduces the time and labor of theadjustment of a fluorescence detector.

[0165] In this embodiment, the case where the nucleotide detector 10 isused as a DNA sensor has been described. However, the nucleotidedetector 10 is used as a RNA sensor by using a group of RNAs, instead ofthe group of DNAs to be detected.

[0166] Furthermore, conventional nucleotide detectors such as DNA chipshave to be disposed of, but in the nucleotide detector 10 of thisembodiment, the substrate and the DNA (or RNA) is fixed firmly via asulfur atom and a gold particle, so that this fixture can be maintainedeven at a temperature of 100° C. Therefore, the nucleotide detector 10can be used repeatedly by dissociating the hybridized DNA from the thiolDNA and washing it away.

[0167] Furthermore, a gold-apoferritin complex in which gold particlesare grown on its outer surface that is obtained in the first embodimentmay be used, instead of the gold-including apoferritin 15 used in thisembodiment. Although the sizes of the gold particles that are grown onthe outer surface of apoferritin are not uniform, but similarly to thegold particles 12 used in this embodiment, the gold particles can bearranged on a substrate with high density and high precision. In thefirst embodiment, when fer-8-Ser-Arg is used, fer-8-Ser-Arg in whichgold particles are grown on the outer surface with very high yield canbe obtained, so that compared to the case where the gold-includingapoferritin 15 is used, the production cost of the nucleotide detector10 can be reduced.

Third Embodiment

[0168] In this embodiment, a nonvolatile memory cell including dotbodies formed by utilizing the gold-including apoferritin produced inthe first Embodiment for a floating gate will be described. It should benoted that the nonvolatile memory cell in this embodiment and the methodfor producing the same are those described in Japanese Laid-Open PatentPublication No. 11-233752.

[0169]FIG. 6 is a cross-sectional view showing the structure of thenonvolatile memory cell utilizing dot bodies for a floating gate. Asshown in FIG. 6, on a p-type Si substrate 21, a polysilicon electrode 26that functions as a control gate, dot bodies 24 that are made of goldfine particles having a particle size of about 6 nm and functions as afloating gate electrode, a gate oxide film 23 that is present betweenthe p-type Si substrate 21 and the floating gate and functions as atunnel insulating film, a silicon oxide film 25 that is present betweenthe control gate and the floating gate and functions as aninterelectrode insulating film for transmitting a voltage of the controlgate to the floating gate are provided. In the p-type Si substrate 21,first and second n-type diffusion layers 27 a and 27 b that function asa source or a drain are formed, and a region between the first andsecond n-type diffusion layers 27 a and 27 b in the p-type Si substrate21 functions as a channel. Furthermore, an element isolation oxide film22 formed by a selection oxidation method or the like for electricalseparation is formed between the memory cell shown in FIG. 6 and amemory cell adjacent thereto. The first and second n-type diffusionlayer 27 a and 27 b are connected to first and second aluminum wiring 31a and 31 b, respectively, via tungsten 30. Although not shown in FIG. 6,the polysilicon electrode 26 and the p-type Si substrate 21 are alsoconnected to aluminum wiring, so that the voltage of each portion of thememory cell is controlled by using the aluminum wiring or the like.

[0170] This memory cell can be formed easily as follows.

[0171] First, the element isolation oxide film 22 enclosing an activeregion is formed by a LOCOS method, and then the gate oxide film 23 isformed on the substrate. Thereafter, the dot bodies 24 are formed overthe entire substrate with the gold-including apoferritin produced in thefirst embodiment. By using the gold-including apoferritin in thisprocess, the process of reducing the dot bodies, which was necessarywhen a conventional apoferritin including a metal oxide was used, can beomitted.

[0172] Next, a silicon oxide film and a polysilicon film to bury the dotbodies 24 are deposited on the substrate by a CVD method.

[0173] Next, the silicon oxide film and the polysilicon film arepatterned so that the silicon oxide film 25 that serves as aninterelectrode insulating film and the polysilicon electrode 26 thatserves as a control gate electrode are formed. Thereafter, impurity ionsare implanted, using a photoresist mask and the polysilicon electrode 26as a mask, so that the first and second n-type diffusion layer 27 a and27 b are formed.

[0174] Then, using known methods, an interlayer insulating film 28 isformed, contact holes 29 are opened in the interlayer insulating film28, tungsten plugs 30 are formed by filling the contact holes 29 withtungsten, and the first and second aluminum wiring 31 a and 31 b areformed.

[0175] The memory cell of this embodiment is provided with a MOStransistor (memory transistor) including the polysilicon electrode 26that functions as the control gate, the first and second n-typediffusion layers 27 a and 27 b that function as the source or the drain,and this memory cell is a nonvolatile memory cell that utilizes thatfact that the threshold voltage of the memory transistor is changed withthe amount of charges accumulated in the dot bodies 24 that function asthe floating gate. This nonvolatile memory cell can be provided with thefunction as a memory storing binary values, but a multivalued memorystoring three or more values can be realized by not only depending onthe presence of charges accumulated in the dot bodies 24, but alsocontrolling the amount of the accumulated charges.

[0176] To erase data, FN (Fowler-Nordhein) current via an oxide film ordirect tunneling current can be utilized.

[0177] To write data, FN (Fowler-Nordhein) current via an oxide film,direct tunneling current or channel hot electron (CHE) implantation canbe utilized.

[0178] According to the nonvolatile memory cell of this embodiment, thefloating gate is made of gold fine particles having a small particlesize so as to function as a quantum dot, so that the amount of theaccumulated charge is small. Therefore, the amount of current for writeand erase can be small, so that a nonvolatile memory cell having a lowpower consumption can be produced.

[0179] Furthermore, in the nonvolatile memory cell of this embodiment,since the sizes of the gold fine particles constituting the floatinggate are uniform, the characteristics at the time of implantation andremoval of charges are uniform among the gold fine particles, so thatcontrol can be performed easily in these operations.

[0180] Furthermore, the dot bodies 24 may be formed continuously whilebeing in contact with each other, that is, may be formed so as toconstitute a film as a whole, or may be formed discretely so that theyare apart from each other. In this embodiment, since the apoferritinincluding gold fine particles is used, such a fine dot body pattern canbe formed easily by subjecting a desired portion of the substrate to atreatment that let the portion hydrophobic, and then arranging theapoferritin or other methods.

[0181] In this embodiment, gold is used as the material of the dotbodies, but instead of this, platinum can be used. Dot bodies made ofplatinum having a uniform diameter of about 6 nm can be formed by usingplatinum-including apoferritin produced in the first embodiment, insteadof the gold-including apoferritin. In this case as well, it isadvantageous that the process of reducing the dot bodies is notnecessary similarly to the case where the gold-including apoferritin isused.

Fourth Embodiment

[0182] In this embodiment, a method for arranging gold particles on asubstrate, utilizing the gold-including apoferritin of the firstembodiment, and using these gold particles as an etching mask will bedescribed.

[0183]FIGS. 7A and 7C are cross-sectional views showing a method forforming microstructures using the gold particles as a mask.

[0184] First, in the process shown in FIG. 7A, the gold-includingapoferritins are arranged in desired positions on a silicon substrate 34in the same manner as in the second embodiment, and then a heattreatment is performed, so that the outer shell made of protein isremoved. Thus, the gold particles 33 having a diameter of about 6 nm areleft on the substrate 34.

[0185] Here, using the apoferritin including gold eliminates thereduction process that is performed when metal oxide-includingapoferritin is used.

[0186] Then, in the process shown in FIG. 7B, ion reactive etching (RIE)is performed with respect to the silicon substrate 34 for 5 minutes witha SF₆ gas, so that the silicon substrate 34 is etched selectively. Thisis because the gold particles 33 are etched with more difficulty thanthe silicon substrate 34.

[0187] Then, in the process shown in FIG. 7C, the gold particles 33 areeventually etched when the etching proceeds further, so that the siliconsubstrate 34 provided with a desired pattern can be obtained. The methodof this embodiment makes it possible to form uniform minutecolumn-shaped pattern whose upper face has a diameter of about 6 nm(hereinafter, referred to as “minute column”) precisely on thesubstrate. In other words, the method of this embodiment makes itpossible to form minute structures having uniform sizes (that is,precise processing of microstructures), which was conventionallydifficult.

[0188] The microstructures formed by the method of this embodiment canbe used as, for example, light-emitting elements utilizing a quantumeffect, which will be described later.

[0189] In this embodiment, the gold particles are used as an etchingmask, but platinum particles can be used instead. For this, in theprocess shown in FIG. 7A, the platinum-including apoferritin of thefirst embodiment can be used, instead of the gold-including apoferritin.

[0190] Employing ferritin including Fe or apoferritin including Ni, Coor the like may eliminate the reduction process as well, depending onthe circumstance. On the other hand, employing the preciousmetal-including apoferritin of this embodiment can eliminate thereduction process in any circumstances.

[0191] In the process shown in FIG. 7A of this embodiment, a heattreatment is used to remove the outer shell of the gold-includingapoferritin, but instead of this, ozonolysis or chemical decompositionwith cyanogen bromide (CNBr) can be used.

Fifth Embodiment

[0192] In a fifth embodiment, a method for producing an opticalsemiconductor device described in Japanese Laid-Open Patent PublicationNo. 08-083940 reported by Eriguchi et al., using the minute columnsformed by the processing method of the fourth embodiment will bedescribed below.

[0193]FIG. 8 is a cross-sectional view of an optical semiconductordevice using semiconductor minute columns whose upper surface has adiameter of 6 nm formed in the fourth embodiment.

[0194] First, the fourth embodiment uses a substrate obtained by forminga p-type well 51 in a part of an n-type silicon, and further forming ann-type well on the p-type well 51. This substrate is processed by themethod of the fourth embodiment, and semiconductor minute columns 42made of n-type silicon are formed with high density.

[0195] Then, the side faces of the semiconductor minute columns arecovered with an insulating layer 43 made of silicon oxide film bythermal oxidation, and then the gaps between the semiconductor minutecolumns 42 are filled with the insulating layer 43 and the end surfacethereof is smoothed.

[0196] Furthermore, the insulating layer on the surface of the smoothedend portion of the semiconductor minute columns 42 of the insulatinglayer 43 is removed, and a transparent electrode 44 is formed.

[0197] The quantitized region Rqa on the silicon substrate 41 on theside is divided from other regions by insulating separation layers 49that have been previously formed. In addition, a side electrode 50penetrating the insulating separation layer 49 has been previouslyformed, and connected to the silicon substrate 41 that functions as alower electrode with respect to the transparent electrode 44, which isthe upper electrode of the semiconductor minute columns 42.

[0198] Thus, an optical semiconductor device is formed, and when avoltage in the forward direction is applied between the transparentelectrode 44 and the side electrode 50, electroluminescence occurs atroom temperature. Furthermore, visible light electroluminescencecorresponding to emission of red, blue and yellow is generated bychanging the carrier implantation voltage.

[0199] According to this embodiment, an optical semiconductor devicehaving a high luminous efficiency, which was conventionally difficult toproduce, can be realized.

Other Embodiments

[0200] In the process of producing the gold-apoferritin complex of thefirst embodiment, a small amount of gold-apoferritin complexes holdinggold particles both on the outer surface and the holding portion can beobtained.

[0201]FIG. 9 is a view showing a gold-apoferritin complex holding goldparticles both on the outer surface and the holding portion. In FIG. 9,the diameter of a first gold particle 61 held in the holding portion isabout 6 nm, and the size of a second gold particle 62 formed on theouter surface of apoferritin has a variation, but it is at least truethat the size is larger than the size of the first gold particle 61included in apoferritin. The first gold particle 61 held in the holdingportion is enclosed by an outer shell 63 of apoferritin.

[0202] The gold-apoferritin complexes are arranged on a siliconsubstrate or the like in the form of a film in such a manner that thesecond gold particle 62 is positioned in an upper portion.

[0203] This substrate is further processed so that a nonvolatile memorycell of a double dot type having the first gold particle 61 and thesecond gold particle 62 as a floating gate can be produced. Thisnonvolatile memory cell is characterized in that the retention time ofdata is long. This is because particles having different sizes aredifferent in how easy they receive or release charges from each other,so that input information can be held in the gold particle that morehardly releases charges. Here, a nonvolatile memory cell having a longretention time can be produced easily by using the gold-apoferritincomplex.

[0204] Furthermore, the apoferritin makes it possible to use goldparticles having a nanometer size as a floating gate, so that a memorycell can be miniaturized.

[0205] In this embodiment, only the gold-apoferritin complex is used,but a complex of other metals and apoferritin can be used in combinationwith the gold-apoferritin complex, so that dots having different levelscan be produced, and therefore a nonvolatile memory having a longretention time can be produced.

[0206] In this embodiment, instead of the apoferritin holding goldparticles both on the outer surface and the holding portion, apoferritinholding platinum particles both on the outer surface and the holdingportion can be used. Alternatively, the apoferritin holding platinumparticles both on the outer surface and the holding portion can be usedin combination with the apoferritin holding gold particles.

[0207] According to the recombinant apoferritin of the present inventionand the method for producing the same, and the preciousmetal-recombinant apoferritin complex and the method for producing thesame, a precious metal atom can be introduced into the apoferritin bymodifying the internal structure using a gene recombination technique,and it is possible to form precious metal particles that can be appliedto various microstructures. Furthermore, the recombinant apoferritin canbe obtained efficiently by using the E. coli and the recombinant genesof the present invention.

1 2 1 504 DNA Artificial Sequence Description of ArtificialSequenceRecombinant DNA of Liver Apoferritin of Equus cebellus 1tattctactg aagtggaggc cgccgtcaac cgcctggtca acctgtacct gcgggcctcc 60tacacctacc tctctctggg cttctatttc gaccgcgacg atgtggctct ggagggcgta 120tgccacttct tccgctgctt ggcggagaag aagcgcaagg gtgccaagtg cctcttgaag 180atgcaaaacc agcgcggcgg ccgcgccctc ttccagagct tgtccaagcc gtcccaggat 240gaatggggta caaccccgga tgccatgaaa gccgccattg tcctggagaa gagcctgaac 300caggcccttt tggatctgca tgccctgggt tctgcccagg cagaccccca tctctgtagc 360ttcttgtcta gccacttcct agacgaggag gtgaaactca tcaagaagat gggcgaccat 420ctgaccaaca tccagaggct cgttggctcc caagctgggc tgggcgagta tctctttgaa 480aggctcactc tcaagcacga ctaa 504 2 167 PRT Artificial Sequence Descriptionof Artificial SequenceRecombinant Liver Apoferritin of Equus cebellus 2Tyr Ser Thr Glu Val Glu Ala Ala Val Asn Arg Leu Val Asn Leu Tyr 1 5 1015 Leu Arg Ala Ser Tyr Thr Tyr Leu Ser Leu Gly Phe Tyr Phe Asp Arg 20 2530 Asp Asp Val Ala Leu Glu Gly Val Cys His Phe Phe Arg Cys Leu Ala 35 4045 Glu Lys Lys Arg Lys Gly Ala Lys Cys Leu Leu Lys Met Gln Asn Gln 50 5560 Arg Gly Gly Arg Ala Leu Phe Gln Asp Leu Gln Lys Pro Ser Gln Asp 65 7075 80 Glu Trp Gly Thr Thr Pro Asp Ala Met Lys Ala Ala Ile Val Leu Glu 8590 95 Lys Ser Leu Asn Gln Ala Leu Leu Asp Leu His Ala Leu Gly Ser Ala100 105 110 Gln Ala Asp Pro His Leu Cys Ser Phe Leu Ser Ser His Phe LeuAsp 115 120 125 Glu Glu Val Lys Leu Ile Lys Lys Met Gly Asp His Leu ThrAsn Ile 130 135 140 Gln Arg Leu Val Gly Ser Gln Ala Gly Leu Gly Glu TyrLeu Phe Glu 145 150 155 160 Arg Leu Thr Leu Lys His Asp 165

What is claimed is:
 1. A recombinant cage-like protein produced by agene recombination technique, comprising: a holding portion that ispresent in an internal portion of the recombinant cage-like protein andcan hold a precious metal particle; and a tunnel-like channel forconnecting the holding portion and an outside of the recombinantcage-like protein.
 2. The recombinant cage-like protein according toclaim 1, wherein the recombinant cage-like protein is apoferritin. 3.The recombinant cage-like protein according to claim 1, wherein theprecious metal particle is gold or platinum.
 4. The recombinantcage-like protein according to claim 1, comprising a first neutral aminoacid that has a smaller molecular size than that of glutamic acid (Glu)and that of aspartic acid (Asp) in positions on an inner surface of thechannel in which a first glutamic acid and a first aspartic acid are tobe present.
 5. The recombinant cage-like protein according to claim 4,wherein the first neutral amino acid is selected from the groupconsisting of serine (Ser), alanine (Ala), and glycine (Gly).
 6. Therecombinant cage-like protein according to claim 1, further comprising abasic amino acid or a second neutral amino acid in a position on aninner surface of the holding portion in which a second glutamic acid isto be present.
 7. The recombinant cage-like protein according to claim6, wherein the basic amino acid or the second neutral amino acid isselected from the group consisting of arginine (Arg), lysine (Lys), andalanine (Ala).
 8. The recombinant cage-like protein according to claim1, wherein at least one cysteine (Cys) substituted for an amino acid ispresent on an inner surface of the holding portion.
 9. The recombinantcage-like protein according to claim 1, comprising a substance having asmaller reduction function than that of cysteine in a position on anouter surface of the recombinant cage-like protein in which cysteine isto be present.
 10. A precious metal—recombinant cage-like proteincomplex comprising a precious metal particle and a recombinant cage-likeprotein, wherein the recombinant cage-like protein comprises a holdingportion that can hold a precious metal particle; and a tunnel-likechannel for connecting the holding portion and an outside of therecombinant cage-like protein.
 11. The precious metal—recombinantcage-like protein complex according to claim 10, wherein the recombinantcage-like protein is apoferritin.
 12. The precious metal—recombinantcage-like protein complex according to claim 10, wherein a gold orplatinum particle is held on an outer surface.
 13. The preciousmetal—recombinant cage-like protein complex according to claim 10,comprising a first neutral amino acid that has a smaller molecular sizethan that of glutamic acid and that of aspartic acid in positions on aninner surface of the channel in which a first glutamic acid and a firstaspartic acid are to be present.
 14. The precious metal—recombinantcage-like protein complex according to claim 13, wherein the firstneutral amino acid is selected from the group consisting of serine,alanine and glycine.
 15. The precious metal—recombinant cage-likeprotein complex according to claim 10, further comprising a basic aminoacid or a second neutral amino acid in a position on an inner surface ofthe holding portion in which a second glutamic acid is to be present.16. The precious metal—recombinant cage-like protein complex accordingto claim 15, wherein the basic amino acid or the second neutral aminoacid is selected from the group consisting of arginine, lysine, andalanine.
 17. The precious metal—recombinant cage-like protein complexaccording to claim 10, wherein at least one cysteine substituted for anamino acid is present on an inner surface of the holding portion.
 18. Arecombinant DNA encoding an amino acid sequence of a recombinantcage-like protein comprising a holding portion that can hold a preciousmetal particle; and a tunnel-like channel for connecting the holdingportion and an outside of the recombinant cage-like protein.
 19. Therecombinant DNA according to claim 18, wherein the recombinant cage-likeprotein is apoferritin.
 20. The recombinant DNA according to claim 18,wherein the precious metal particle is gold or platinum.
 21. Therecombinant DNA according to claim 18, comprising a first neutral aminoacid that has a smaller molecular size than that of glutamic acid andthat of aspartic acid in positions on an inner surface of the channel inwhich a first glutamic acid and a first aspartic acid are to be present.22. The recombinant DNA according to claim 21, wherein the first neutralamino acid is selected from the group consisting of serine, alanine andglycine.
 23. The recombinant DNA according to claim 18, furthercomprising a basic amino acid or a second neutral amino acid in aposition on an inner surface of the holding portion in which a secondglutamic acid is to be present.
 24. The recombinant according to claim23, wherein the basic amino acid or the second neutral amino acid isselected from the group consisting of arginine, lysine, and alanine. 25.The recombinant DNA according to claim 18, wherein at least one cysteinesubstituted for an amino acid is present on an inner surface of theholding portion.
 26. A method for producing a recombinant cage-likeprotein, comprising the step (a) of substituting a first glutamic acidand a first aspartic acid that are positioned on an inner surface of achannel with a first neutral amino acid having a smaller molecular sizethan that of glutamic acid and that of aspartic acid.
 27. The method forproducing a recombinant cage-like protein according to claim 26, whereinthe cage-like protein is apoferritin.
 28. The method for producing arecombinant cage-like protein according to claim 26, wherein in the step(a), the first neutral amino acid is selected from the group consistingof serine, alanine, and glycine.
 29. The method for producing arecombinant cage-like protein according to claim 26, further comprisingthe step (b) of substituting a second glutamic acid present on an innersurface of the holding portion that is inside the recombinant cage-likeprotein with a basic amino acid or a second neutral amino acid.
 30. Themethod for producing a recombinant cage-like protein according to claim26, wherein in the step (b), the basic amino acid or the second neutralamino acid is selected from the group consisting of arginine, lysine andalanine.
 31. The method for producing a recombinant cage-like proteinaccording to claim 26, further comprising the step (c) of substitutingat least one amino acid positioned on an inner surface of the holdingportion with cysteine.
 32. The method for producing a recombinantcage-like protein according to claim 26, further comprising the step (d)of replacing at least one cysteine positioned on an outer surface of therecombinant cage-like protein by a substance having a smaller reductionfunction than that of cysteine.
 33. A method for producing a preciousmetal-recombinant cage-like protein complex comprising the steps: (a)mixing a precious metal complex ion solution and a recombinant cage-likeprotein solution to form a precious metal-recombinant cage-like proteincomplex, and (b) passing a solution containing the preciousmetal-recombinant cage-like protein complex prepared in the step (a)through a gel filtration column to purify the precious metal-recombinantcage-like protein complex.
 34. The method for producing a preciousmetal-recombinant cage-like protein complex according to claim 33,wherein the precious metal in the step (a) is gold or platinum.